Glaucoma is a leading cause of irreversible blindness in the world, and it affects more than 285,000 U.S. veterans. Elevated intraocular pressure (IOP) is associated with both development and progression of primary open angle glaucoma, and IOP is the only modifiable risk factor for the disease. However, the mechanisms causing elevated IOP remain poorly understood. Trabecular meshwork cells are critical to maintaining a normal aqueous fluid flow and intraocular pressure. In other tissues such as the kidney, bones and cartilage, the primary cilia play a mechanosensory role in fluid flow. Here, we further explore the potential for cilia of trabecular meshwork cells to serve a mechanosensory role in the eye. Indeed, our previous findings demonstrate that phosphoinositide lipids within primary cilium are an important regulator of eye pressure. In the primary cilium, inositol phosphatase OCRL interacts with TRPV4, a mechanosensory channel that lowers IOP in mice when TRPV4 is stimulated. Previous studies have established that phosphoinositides regulate TRPV4 activity. We hypothesize that phosphoinositide signaling within the cilia of the trabecular meshwork cells regulates aqueous outflow via TRPV4 channels. In this application, we propose to employ novel optogenetic techniques to selectively direct protein translocation and activation of ciliary inositl kinase/phosphatase to modulate TRPV4 activity and IOP. In Aim 1, we will determine whether alterations in PIP2 dephosphorylation via OCRL affect ciliary outflow facility and IOP in mice by optogenetic stimulation of inositol phosphatase OCRL. We will then assess whether optical stimulation of ectopically expressed OCRL can rescue a steroid-induced glaucoma (SIG) mouse model. We expect that the activation of TRPV4 channels via light-responsive OCRL in the anterior chamber of mouse eye will lower IOP. In Aim 2, we will determine whether PIP2 phosphorylation via PI3K activation will affect ciliary outflow facility and IOP in wild-type mice. We anticipate that optogenetic stimulation of PI3K signaling will increase IOP and reduce outflow facility. We will then determine whether small molecule inhibition of PI3K decreases IOP in the steroid-induced glaucoma mouse model. In Aim 3, we will determine whether steroid-induced glaucoma patients exhibit changes in their trabecular meshwork cell membrane phosphoinositide content. We will also compare phosphoinositide levels in cultured trabecular meshwork cells from patients undergoing trabeculectomy surgery to normal aged- matched cadaveric controls. The proposed research is expected to have a significant impact on the understanding of mechanosensation of eye pressure in mammals, thereby facilitating the discovery of new therapies for open angle glaucoma and other forms of glaucoma. Ultimately, the knowledge obtained from this study may allow for the development of new therapeutic strategies in glaucoma that will benefit our veterans.